The structure and the binding reactions of the thyroxine transport proteins of serum have been investigated further. The 20 N-terminal amino acids of thyroxine-binding globulin (TBG) were sequenced, and appear to be unique to this protein. Totally deglycosylated TBG retains the ability to bind thyroxine. Affinity labeling showed that tyrosine and lysine residues are constituents of the binding surface. By the use of spin-labeled analogs of thyroxine, it was shown that the size and shape of the binding sites in albumin, PA and TBG are different. The negative cooperativity observed in thyroxine binding to prealbumin appears to be due to an allosteric effect caused by binding of the first hormone molecule to one of the two identical binding sites.